STRUCTURAL AND MOLECULAR BASES FOR THE CONSTITUTIVE ACTIVITY OF IKACH IN CHRONIC ATRIAL FIBRILLATION

Background: A hallmark of remodeling in chronic atrial fibrillation (AF) is action potential duration (APD) shortening. It has been suggested that in chronic AF: (1) the acetylcholine-sensitive inward rectifier potassium current is constitutively active (ca-IKACh); (2) ca-IKACh is due to interaction between Kir3.1 (a molecular correlate of IKACh) and protein kinase C epsilon (PKCe); and (3) oxidative stress (OS) is increased. However, the structural and molecular bases of ca-IKACh and its possible role in APD shortening and AF maintenance remain poorly understood. We hypothesized that OS phosphorylates Kir3.1 at residue S185 via PKCe, leading to ca-IKACh and contributing to APD shortening and AF perpetuation. Methods and Results: We tested our hypothesis using molecular, electrophysiologic, and numerical approaches. We induced OS with 100 μM H2O2 for 1 hour. In HEK cells transfected with Kir3.1/4, OS increased reactive oxygen species as assayed by dihydroethidium fluorescence. In addition, PKCe translocated to the membrane, Kir3.1 showed phosphorylation at S185, and IKACh increased from –16.1 5.1 pA/pF (n 1⁄4 3) to –34.2 3.8 pA/pF (n 1⁄4 6, Po.01). PKCe silencing prevented OS-induced phosphorylation of Kir3.1 and IKACh increase. In mouse atrial myocytes, OS shortened APD90 from 98 13 ms (n 1⁄4 6) to 37.9 5.3 ms (n 1⁄4 13, Po.01). Tertiapin Q, a selective IKACh blocker, significantly prolonged APD90 in stressed but not unstressed myocytes. In sheep atria with tachypacing-induced chronic AF, phospho-Kir3.1 increased 2.45-fold in chronic AF (n 1⁄4 4) vs sham (n 1⁄4 4, Po.05). Optical maps showed that tertiapin Q progressively reduced chronic AF dominant frequency until sinus rhythm resumed. Molecular dynamics simulations using the chimeric Kir3.1 crystal structure suggested that the region containing S185 was a switch that initiated conformational changes resulting in channel opening upon phosphorylation. Finally, purified chimeric Kir3.1 was phosphorylated in vitro by recombinant activated PKCe. Conclusions: Arrhythmogenic ca-IKACh is due in part to OS-mediated PKCe activation and Kir3.1 phosphorylation at residue S185. These results shed new light on the complex ionic mechanisms of AF and may help in the development of new anti-AF agents.